Macrophages enhance Vegfa-driven angiogenesis in an embryonic zebrafish tumour xenograft model

Tumour angiogenesis has long been a focus of anti-cancer therapy; however, anti-angiogenic cancer treatment strategies have had limited clinical success. Tumour-associated myeloid cells are believed to play a role in the resistance of cancer towards anti-angiogenesis therapy, but the mechanisms by which they do this are unclear. An embryonic zebrafish xenograft model has been developed to investigate the mechanisms of tumour angiogenesis and as an assay to screen anti-angiogenic compounds. In this study, we used cell ablation techniques to remove either macrophages or neutrophils and assessed their contribution towards zebrafish xenograft angiogenesis by quantitating levels of graft vascularisation. The ablation of macrophages, but not neutrophils, caused a strong reduction in tumour xenograft vascularisation and time-lapse imaging demonstrated that tumour xenograft macrophages directly associated with the migrating tip of developing tumour blood vessels. Finally, we found that, although macrophages are required for vascularisation in xenografts that either secrete VEGFA or overexpress zebrafish vegfaa, they are not required for the vascularisation of grafts with low levels of VEGFA, suggesting that zebrafish macrophages can enhance Vegfa-driven tumour angiogenesis. The importance of macrophages to this angiogenic response suggests that this model could be used to further investigate the interplay between myeloid cells and tumour vascularisation

Effect of wortmannin and phorbol ester on Paramecium fluid-phase uptake in the presence of transferrin

The kinetics of the uptake of the fluid phase marker Lucifer Yellow (LY), and its alteration by wortmannin, an inhibitor of phosphatidylinositol-3 kinase (PI-3K), and the PKC modulators: GF 109203 X, an inhibitor, and phorbol ester, an activator was studied in eukaryotic model Paramecium aurelia. Spectrophotometric quantification of LY accumulation was performed in the presence or absence of transferrin, a marker of receptor-mediated endocytosis. Internalization of LY showed a curvilinear kinetics: the high initial rate of LYuptake (575 ng LY/ mg protein /hr) decreased almost 5-fold within 15 min, reaching plateau at 126 ng/ mg protein /hr. Transferrin induced a small increase (7.5%) in the fluid phase uptake rate (after 5 min) followed by a small decrease at longer incubation times. Lucifer Yellow and transferrin (visualized by streptavidin– FITC) were localized in Paramecium by 3-D reconstruction by confocal microscopy. LY showed a scattered, diffuse fluorescence typical of fluid phase uptake whereas transferrin accumulated in membrane-surrounded endosomes. Wortmannin did not affect LY accumulation but decreased it when transferrin was present in the incubation medium. This suggests an effect on the transferrin uptake pathway, presumably on the stage of internalization in “mixing” endosomes to which transferrin and LY were targeted. Phorbol ester diminished LY accumulation by 22% and this effect persisted up to 25 min of incubation. PKC inhibitor did not affect LY uptake. However, in the presence of transferrin, the LY uptake increased within the first 15 minutes followed by a rapid 20% decrease in comparison to the control. Such an effect of PKC modulators suggests that PMA action on fluid phase uptake is not directly mediated by PKC

Site‐directed mutagenesis, in vivo electroporation and mass spectrometry in search for determinants of the subcellular targeting of Rab7b paralogue in the model eukaryote Paramecium octaurelia

Protein products of the paralogous genes resulting from the whole genome duplication may acquire new function. The role of post‐translational modifications (PTM) in proper targeting of Paramecium Rab7b paralogue – distinct from that of Rab7a directly involved in phagocytosis ‐ was studied using point mutagenesis, proteomic analysis and double immunofluorescence after in vivo electroporation of the mutagenized protein. Here we show that substitution of Thr200 by Ala200 resulted in diminished incorporation of [P32] by 37.4% and of 32 [C14–]UDP‐glucose by 24%, respectively, into recombinant Rab7b_200 in comparison to the non‐mutagenized control. Double confocal imaging revealed that Rab7b_200 was mistargeted upon electroporation into living cells contrary to non‐ mutagenized recombinant Rab7b correctly incorporated in the cytostome area. We identified the peptide ion at m/z=677.63+ characteristic for the glycan group attached to Thr200 in Rab7b using nano LC‐MS/MS and comparing the peptide map of this protein with that after deglycosylation with the mixture of five enzymes of different specificity. Based on the mass of this peptide ion and quantitative radioactive assays with [P32]and  [C14‐]UDP‐ glucose, the suggested composition of the adduct attached to Thr200 might be (Hex)1(HexNAc)1(Phos)3 or (HexNAc)1 (Deoxyhexose)1 (Phos)1 (HexA)1. These data indicate that PTM of Thr200 located in the hypervariable C‐region of Rab7b in Paramecium is crucial for the proper localization/function of this protein. Moreover, these proteins differ also in other PTM: the number of phosphorylated amino acids in Rab7b is much higher than in Rab7a.   </p